Tyrosinase Lab Report

The results of the three-part experiment provide a deeper knowledge about the factors that influence the rate of the reaction of the enzyme activity and how the factors influence the structure or function of the enzyme.

J. Moldovan & B. Nilson, (2010), Lab 4 – Enzyme Kinetics, UBCO BIOL/BIOC 393, UBC Vista accessed Monday, November 8th, 2010.

From the stock substrate solution of 2.5 mM, each group serially diluted at least one different substrate concentration for a total of four different substrate concentrations to be investigated: 1.25 mM, 1.0 mM, 0.75 mM, 0.25 mM. The enzyme concentration was kept constant at 2.0 mM while experimenting on the affect of varying enzyme concentration on the rate and product formation of ONP. Enough 2.0 mM enzyme solution was prepared in the previous part of the project to supply this assay. Using similar procedure to collect absorbance data as the first part, 0.5 mL of 2.0 mM enzyme concentration was placed into the cuvette and used to calibrate the spectrometer at 420 nm. Data was then started, with the immediate addition of 0.5 mL of varying substrate concentrations. Each varying substrate concentration was split between the team and run for a total of 10 minutes, with the exception of the 1.25 mM run. Upon completion, data from each varying substrate concentration was copied to a single Excel sheet and used to produce an absorbance vs. time graph, product formation vs. time graph, Michaelis Menten plot, and Lineweaver-Birk plot. This analysis was used to calculate the V0,Vmax, and Km for β-Galactosidase

Background and Introduction: Enzymes are proteins that process substrates, which is the chemical molecule that enzymes work on to make products. Enzyme purpose is to increase the rate of activity and speed up chemical reaction in a form of biological catalysts. The enzymes specialize in lowering the activation energy to start the process. Enzymes are very specific in their process, each substrate is designed to fit with a specific substrate and the enzyme and substrate link at the active site. The binding of a substrate to the active site of an enzyme is a very specific interaction. Active sites are clefts or grooves on the surface of an enzyme, usually composed of amino acids from different parts of the polypeptide chain that are brought together in the tertiary structure of the folded protein. Substrates initially bind to the active site by noncovalent interactions, including hydrogen bonds, ionic bonds, and hydrophobic interactions. Once a substrate is bound to the active site of an enzyme, multiple mechanisms can accelerate its conversion to the product of the reaction. But sometimes, these enzymes fail or succeed to increase the rate of action because of various factors that limit the action. These factors can be known as temperature, acidity levels (pH), enzyme and/or substrate concentration, etc. In this experiment, it will be tested how much of an effect

However, the rate of reaction only increases for a certain period of time until there is lesser substrate molecules than the enzyme molecules. The increase of enzyme concentration does not have effect if there are lesser substrate molecules than enzyme molecules initially.

Graphs will be used with the enzyme alkaline phosphatase of the unknown in the enzyme solution to Determine Km and Vmax

This is because the more neutral the pH the faster enzymes can catalyse reactions. If there is an excess of H⁺ or OH⁻ ions then the shape of the protein alters, thus slowing down catalysis. The graph shows this as pH7 is neutral and has the highest reaction rates, while pH3 and pH11, acidic and basic, respectively, both have fairly low reaction rates. pH5 and pH9 are both in the middle because they are slightly acidic, so they have a lower reaction rate than pH7, but a higher reaction rate than pH3 and pH11. Describe the relationship between substrate concentration and the initial reaction rate of an enzyme-catalyzed reaction.

However, temperature difference can affect the experiment. To prevent this, I will conduct the experiment at the same time of day if more than one day is needed. This is because it is generally colder in the mornings and evening than the afternoon.

[An active site can be altered by a non-competitive enzyme which encircles the enzyme and alters the shape of the active site which could be very dangerous.]

Organisms cannot depend solely on spontaneous reactions for the production of materials because they occur slowly and are not responsive to the organism's needs (Martineau, Dean, et al, Laboratory Manual, 43). In order to speed up the reaction process, cells use enzymes as biological catalysts. Enzymes are able to speed up the reaction through lowering activation energy. Additionally, enzymes facilitate reactions without being consumed (manual,43). Each enzyme acts on a specific molecule or set of molecules referred to as the enzyme's substrate and the results of this reaction are called products (manual 43). As a result, enzymes promote a reaction so that substrates are converted into products on a faster pace (manual 43). Most enzymes are proteins whose structure is determined by its sequence of its amino acids. Enzymes are designed to function the best under physiological conditions of PH and temperature. Any change of these variables that change the conformation of the enzyme will destroy or enhance enzyme activity(manual, 43).

Enzymes are natural catalysts that work from the ability to increase the rate of reaction by decreasing the activation energy of a reaction. (Blanco, Blanco 2017) An enzyme can do this 10^8- to 10^10 fold, sometimes even 10^15 fold. (Malacinsk, Freifelder 1998) The substrate will momentarily bind with the enzyme making the enzyme-substrate complex, of which the shape of the substrate is complimentary to the shape of the active site on the enzyme it is binding with. There are two main theories as to how an enzymes and substrates interact, the lock-and-key model and induced fit theory. The lock-and-key model suggests that the enzyme has a specific shape that fits the substrate and only that substrate. The induced fit theory says the active site and substrate are able to change shape or distort for the reaction to take place with (Cooper,

reaction mechanism. Therefore, if an enzyme has a high KM value, then it has a relatively low affinity for its substrate since it requires a higher substrate concentration to reach its vmax. In contrast, a low KM value corresponds to a high substrate affinity. The vmax value corresponds to the rate of reaction at which the enzyme is completely saturated with substrate. Therefore, this vmax value reflects how fast an enzyme can catalyze a given reaction.

The independent variable in this investigation is pH. Each individual enzyme has it’s own pH characteristic. This is because the hydrogen and ionic bonds between –NH2 and –COOH groups of the polypeptides that make up the enzyme, fix the exact arrangement of the active site of an enzyme. It is crucial to be aware of how even small changes in the

An enzyme is a catalyst. Catalysts are known for speeding up the rate of reactions by lowering the activation energy of the biochemical reaction. (Reece et al., 2011)

Enzymes are biological catalysts, which means it decreases activation energy in reactions. The lower activation energy in a reaction, the faster the reaction rate. Many enzymes alter their shape when they bind to the activation site. This is called induced fit, meaning for the enzyme to work to its full potential it has to change shape to binding substrate. The location of enzyme’s activation site is on the surface of the enzyme, where the binding of substrates take place. Enzyme activity can be influenced by a variety of environmental factors. If the concentration of enzyme is low, and there is a great deal of substrate, then increasing enzyme concentration results in more molecules available to convert substrates to products. Thus, increasing enzyme concentration can increase reaction rate. If substrate concentrations are low, and many of the existing enzymes are idle because of a lack of substrate, then adding enzyme will have no effect on reaction rate. Enzyme concentration affects the enzyme activity, because the more enzyme concentration the faster the reaction rate, until it hits it’s limiting factor. When substrate concentration is increased, it also increases rate of reaction. Temperature plays an important